ORIGINAL PAPER An inducible NADPH–cytochrome P450 reductase from Picrorhiza kurrooa — an imperative redox partner of cytochrome P450 enzymes Wajid Waheed Bhat & Satiander Rana & Niha Dhar & Sumeer Razdan & Shahzad A. Pandith & Ram Vishwakarma & Surrinder K. Lattoo Received: 9 October 2013 /Revised: 31 December 2013 /Accepted: 27 January 2014 /Published online: 13 February 2014 Abstract Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glyco- sides called picrosides. Although chemical profile and phar- macological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH–cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were deter- mined. Specific activity, V max and K m of PkCPR were found to be 5.8±0.05 μmol min -1 mg -1 , 8.1±0.12 μmol min -1 mg -1 and 7.8 μM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collect- ed at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reac- tions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa. Keywords Elicitors . Functionalcharacterization . Homology modelling . Picrorhiza kurrooa . Picrosides . NADPH– cytochrome P450 reductase Abbreviations IPTG Isopropyl-β-D-thiogalactopyranoside ORF Open reading frame CPR Cytochrome P450 reductase RACE Rapid amplification of cDNA ends RT-PCR Reverse transcription-polymerase chain reaction MeJA Methyl jasmonate SA Salicylic acid 2,4-D 2,4-Dicholorophenoxy acetic acid SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis TSS Transcriptional start site UTR Untranslated region Introduction Cytochrome P450s (CYP450s) are heme thiolate-proteins that catalyse extremely diverse variety of reactions like hydroxyl- ations, dealkylations, sulphoxidations, epoxidations, reduc- tive dehalogenations, peroxidations and various types of isomerization for the synthesis of a suite of primary and Electronic supplementary material The online version of this article (doi:10.1007/s10142-014-0362-7) contains supplementary material, which is available to authorized users. W. W. Bhat : S. Rana : N. Dhar : S. Razdan : S. A. Pandith : S. K. Lattoo (*) Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, India e-mail: sklattoo@iiim.ac.in R. Vishwakarma Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, India Funct Integr Genomics (2014) 14:381–399 DOI 10.1007/s10142-014-0362-7 # Springer-Verlag Berlin Heidelberg 2014